We report here the identification of a T-cell lineage protein designated AGAPE (ä-gä-pa; Another Grb2-Associated Protein Enigma). AGAPE contains a proline rich sequence that associates with the adaptor protein Grb2. It lacks any consensus catalytic domains but contains a nuclear localization sequence required for its translocation to the nucleus. AGAPE deficient mice were generated by homologous recombination in ES cells. AGAPE-/- mice have normal numbers of DN and DP thymocytes but markedly reduced numbers of CD4 SP and CD8 SP thymocytes and pheripheral T cells. Positive selection is dramatically reduced in both class II- and class I-restricted TCR transgenic AGAPE-/- mice. The proportion of CD4+CD8lo thymocytes was reduced in AGAPE-/- MHCII-/- mice suggesting that AGAPE plays and important role at the DP to SP transition stage. Interestingly, TCR induced activation responses are unaffected in AGAPE-/- thymocytes indicating that the developmental block is not due to a defect in signaling downstream of the TCR? Although DP thymocytes from AGAPE-/- mice show enhanced susceptibility to antibody-induced cell death, the introduction of the Bcl-2 transgene did not rescue T cell development. Expression of the lineage specific transcription factors ThPok and Gata-3 (but not Tox or Runx1/3) was reduced in CD69+ thymocytes purified from AGAPE-/- mice. However, enforced expression of ThPok or Gata-3 did not rescue CD4 SP development in AGAPE-/- mice. Thus, AGAPE is a new T cell specific nuclear protein that has a critical function during late thymocyte selection prior to CD4/CD8 lineage specification.